INH1

INH1 is a potent inhibitor of Hec1/Nek2 [1].

Hec1 is an oncogene that involved in spindle checkpoint signaling and is overexpressed in many human cancers. Nek2 is serine/threonine-protein kinase that phosphorylates Hec1, which is critical for its mitotic function and cell survival [1].

INH1 is a potent inhibitor of Hec1/Nek2 via directly binding to Hec1. INH1 (1 and 20 µM) inhibited the ability of chip-immobilized Hec1 binding to free Nek2 by 39% and 55% but didn’t affect immobilized Nek2 binding to free Hec1, which suggested that INH1 directly bound to Hec1. In the lysate from cells treated with INH1 (25 µM), the coimmunoprecipitate of Hec1 with Nek2 was inhibited, suggesting that INH1 disrupted the Hec1/Nek2 complex. INH1 (25 µM) significantly reduced cellular Nek2 protein level by 80-90% in a time- and dose-dependent way and reduced kinetochore-bound Hec1 pool by 55%. In human breast cancer cell lines, INH1 inhibited cell proliferation with GI50 values of 10-21 µM. In HeLa cells, INH1 increased the mitotic index by 2-fold and induced mitotic abnormalities [1].

In nude mice xenografted breast cancer, INH1 inhibited tumor growth [1].

Reference:
[1].  Wu G, Qiu XL, Zhou L, et al. Small molecule targeting the Hec1/Nek2 mitotic pathway suppresses tumor cell growth in culture and in animal. Cancer Res, 2008, 68(20): 8393-8399.

The F1-ATPase inhibitor Inh1 (IF1) affects suppression of mtDNA loss-lethality in Kluyveromyces lactis.[Pubmed:17286560]

FEMS Yeast Res. 2007 Aug;7(5):665-74.

Loss of mtDNA by the petite-negative yeast Kluyveromyces lactis is lethal (rho(o)-lethality). However, mutations in the alpha, beta and gamma subunits of F(1)-ATPase can suppress lethality by increasing intramitochondrial hydrolysis of ATP. Increased hydrolysis of ATP can also occur on inactivation of INH1, the natural inhibitor of F(1)-ATPase. However, not all strains of K. lactis show suppression of rho(o)-lethality on inactivation of INH1. Genetic analysis indicates that one or more alleles of modifying factors are required for suppression. Papillae showing enhanced resistance to ethidium bromide (EB) in INH1 disruptants have mutations in the alpha, beta and gamma subunits of F(1)-ATPase. Increased growth of double mutants on EB has been investigated by disruption of INH1 in previously characterized atp suppressor mutants. Inactivation of INH1, with one exception, results in better growth on EB and increased F(1)-ATPase activity, indicating that suppression of rho(o)-lethality is not due to atp mutations preventing INH1 from interacting with the F(1)-complex. By contrast, in suppressor mutants altered in Arg435 of the beta subunit, disruption of INH1 did not change the kinetic properties of F(1)-ATPase or alter growth on EB. Consequently, Arg435 appears to be required for interaction of INH1 with the beta subunit. In a previous study, a mex1-1 allele was found to enhance mgi(atp) expression. In accord with results from double mutants, it has been found that mex1-1 is a frameshift mutation in INH1 causing inactivation of INH1p.

Formation of the yeast F1F0-ATP synthase dimeric complex does not require the ATPase inhibitor protein, Inh1.[Pubmed:12167646]

J Biol Chem. 2002 Oct 18;277(42):39289-95.

The yeast F1F0-ATP synthase forms dimeric complexes in the mitochondrial inner membrane and in a manner that is supported by the F0-sector subunits, Su e and Su g. Furthermore, it has recently been demonstrated that the binding of the F1F0-ATPase natural inhibitor protein to purified bovine F1-sectors can promote their dimerization in solution (Cabezon, E., Arechaga, I., Jonathan P., Butler, G., and Walker J. E. (2000) J. Biol. Chem. 275, 28353-28355). It was unclear until now whether the binding of the inhibitor protein to the F1 domains contributes to the process of F1F0-ATP synthase dimerization in intact mitochondria. Here we have directly addressed the involvement of the yeast inhibitor protein, INH1, and its known accessory proteins, Stf1 and Stf2, in the formation of the yeast F1F0-ATP synthase dimer. Using mitochondria isolated from null mutants deficient in INH1, Stf1, and Stf2, we demonstrate that formation of the F(1)F(0)-ATP synthase dimers is not adversely affected by the absence of these proteins. Furthermore, we demonstrate that the F1F0-ATPase monomers present in su e null mutant mitochondria can be as effectively inhibited by INH1, as its dimeric counterpart in wild-type mitochondria. We conclude that dimerization of the F1F0-ATP synthase complexes involves a physical interaction of the membrane-embedded F0 sectors from two monomeric complexes and in a manner that is independent of inhibitory activity of the INH1 and accessory proteins.

Enhancement of pyruvate productivity by inducible expression of a F(0)F(1)-ATPase inhibitor INH1 in Torulopsis glabrata CCTCC M202019.[Pubmed:19761804]

J Biotechnol. 2009 Oct 26;144(2):120-6.

The aim of this study is to establish a controllable intracellular ATP content regulation system applied to the enhancement of pyruvate production in Torulopsis glabrata. The INH1 gene, which encodes a F(0)F(1)-ATPase inhibitor from Saccharomyces cerevisiae, was expressed under a copper ion inducible promoter in the pyruvate producer Torulopsis glabrata CCTCC M202019. The resultant strain was designated as T. glabrata INH1. The induction efficiency was measured by the inducible expression of an enhanced green fluorescence protein. The copper inducible INH1 gene could control the intracellular ATP content (24 h) in an extensive range between 0.192 mmol/mg protein and 0.642 mmol/mg protein in a flask culture. With T. glabrataINH1, induction with 30 microM of Cu(2+) at 12 h in a 3 L fermentor improved pyruvate yield from glucose on biomass by 29% and its yield by 20%, respectively.

Small molecule targeting the Hec1/Nek2 mitotic pathway suppresses tumor cell growth in culture and in animal.[Pubmed:18922912]

Cancer Res. 2008 Oct 15;68(20):8393-9.

Hec1 is a conserved mitotic regulator critical for spindle checkpoint control, kinetochore functionality, and cell survival. Overexpression of Hec1 has been detected in a variety of human cancers and is linked to poor prognosis of primary breast cancers. Through a chemical genetic screening, we have identified a small molecule, N-(4-[2,4-dimethyl-phenyl]-thiazol-2-yl)-benzamide (INH1), which specifically disrupts the Hec1/Nek2 interaction via direct Hec1 binding. Treating cells with INH1 triggered reduction of kinetochore-bound Hec1 as well as global Nek2 protein level, consequently leading to metaphase chromosome misalignment, spindle aberrancy, and eventual cell death. INH1 effectively inhibited the proliferation of multiple human breast cancer cell lines in culture (GI(50), 10-21 micromol/L). Furthermore, treatment with INH1 retarded tumor growth in a nude mouse model bearing xenografts derived from the human breast cancer line MDA-MB-468, with no apparent side effects. This study suggests that the Hec1/Nek2 pathway may serve as a novel mitotic target for cancer intervention by small compounds.